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Introduction: LC-MS Analysis of Biological Samples

Phospholipids are major components of all cell membranes; and phospholipid contamination has been identified as one of the principal causes of ion-suppression when performing LC-MS analysis of small molecules in biological samples. They also buildup on analytical columns, elute unpredictably in downstream analyses and increase the need for sample reprocessing. Due to their impact on data quality, many labs want to remove phospholipids prior to LC-MS analysis. Business needs, however, do not allow for an increase in sample prep costs and processing times. Labs often find themselves forced to make economic decisions at the expense of data quality.

Not only does phospholipid-induced ion suppression impact LC-MS data accuracy and reproducibility, but phospholipids also buildup on analytical columns and increase the frequency of column replacement. Unfortunately, traditional sample prep methods, such as protein precipitation (PPT) and liquid-liquid extraction (LLE), do not remove phospholipids. In addition, SPE which can be both costly and time-consuming, only provides nominal phospholipid removal.

Phospholipid removal products efficiently remove phospholipids and reduce ion-suppression; however, they also double sample prep cost and processing time as compared to standard protein PPT. Furthermore, the hydrophobic retention mechanisms employed in most phospholipid removal products are problematic when working with hydrophobic analytes, requiring analysts to perform method development to find a balance between effective phospholipid removal and recovery of the hydrophobic analyte(s).

Solution

HybridSPE-PLus provides a simple, proven method for the removal of both precipitated proteins and phospholipids. Unlike competitive phospholipid removal products, its unique Lewis acid/base retention mechanism can provide efficient phospholipid removal and good recoveries of both hydrophobic and hydrophilic analytes. Studies conducted to determine the number of LC-MS injections that can be obtained for samples processed via standard protein precipitation, and samples processed with HybridSPE-PLus prior to column replacement showed a 10-fold difference in replacement frequency between the two sample prep methods. Samples processed via standard protein precipitation showed a >20% drop in analyte response after approximately100 injections; whereas, the same samples processed via HybridSPE-PLus showed minimal decline in analyte response at >1,000 injections.

Benefits

Reduce overall cost

Reduce overall processing time

Improve LC-MS reproducibility

Decrease need for sample reprocessing

By implementing HybridSPE-PLus for sample prep of all biological samples prior to LC-MS analysis, labs can offset increased sample prep costs and processing times associated with phospholipid removal with a 10-fold reduction in analytical column costs and time associated with column replacement, conditioning and equilibration.

Figure 1. Overall Cost per Analysis

Assuming column costs of $500 per column, sample prep costs of ~$1 per sample for protein precipitation and ~$2 per sample for HybridSPE-PLus plates, total costs of processing 1,000 samples would be $6,000 for samples processed via standard protein precipitation (with column replacement every 100 injections) and $2,500 for samples processed via HybridSPE-PLus (with column replacement every 1,000 injections). That is a difference of $3.50 per sample.

Figure 2. Time Savings per Analysis

Similarly, although the standard HybridSPE-PLus plate methodology adds up to 5 minutes to the sample prep time required for every ~100 samples as compared to standard protein precipitation, this is greatly offset by the reduction in time spent replacing, conditioning and equilibrating columns. For every 1,000 samples processed via HybridSPE-PLus plates, an additional 50 minutes is required for sample prep plus 20 minutes for column replacement, conditioning and equilibration. Meanwhile, 200 minutes are required for column replacement, conditioning and equilibration when processing those same 1,000 samples using standard protein precipitation. Overall processing time per sample is reduced by ~0.13 minutes per sample when HybridSPE-PLus plates are used for sample prep. Implementation of HybridSPE-PLus plates therefore provides a solution that allows labs to achieve both their economic and data quality objectives.

All Your LC-MS Needs In One Place

In addition to our HybridSPE-Phospholipid technology, we provide the following premier selection of proven tools and consumables for your entire sample prep and LC-MS workflows.